PROJECT SUMMARY The major challenges for developing primary prevention strategies for childhood asthma are the early identification of modifiable risk factors (e.g., epigenome, IgE sensitization, overweight/ obesity) and the heterogeneity of asthma. The overarching objective of this R01 project is to investigate the role of blood DNA methylation (DNAm) during infancy in the development of three outcomes: IgE sensitization, overweight/obesity (and adiposopathy), and eventually asthma in two complementary multicenter prospective cohort studies. The 35th Multicenter Airway Research Collaboration (MARC-35) study (U01AI087881) is an ongoing 17-center cohort study of 921 infants hospitalized for bronchiolitis ? a population at high risk for asthma. Another ongoing cohort study, MARC-43 (UG3/UH3 OD023253), includes 600 healthy infants. These racially/ethnically-diverse cohorts (52% African American or Hispanic) are truly complementary, with participants undergoing similar procedures (e.g., specific IgE and cytokine measurements) at similar ages (e.g., infancy, ages 3 and 6 years). Follow-up includes biannual interviews and medical records to age 6+ years, with ~90% follow-up to date. Participants are undergoing in-person examination at age 6 years for asthma phenotyping. The present R01 project would extend these large well-characterized cohorts by profiling the blood genome-wide DNAm in 1,521 infants, and then examining their relations to the development of the three main outcomes: IgE sensitization, overweight/obesity, and asthma. In Aim 1, we will identify the associations of infant blood DNAm signature with the risk of developing asthma, including its phenotypes. We will also investigate the longitudinal changes of these DNAm from infancy to age 3 years, and their relations to asthma risk. In Aim 2, we will examine the relations of infant blood DNAm signature with the risk of developing IgE sensitization and of overweight/obesity (and adiposopathy). In Aim 3, we will determine the role of IgE sensitization and of overweight/ obesity in the link between infant DNAm and asthma. Our pilot data lend compelling support to these hypotheses. In Aim 4, we will also integrate the available multi-omics data to further define the mechanisms that underlie DNAm signatures identified in Aims 1-3. We will replicate our findings in 963 children from a harmonized birth cohort ? the Boston Birth Cohort. The R01 project will provide a unique opportunity to define the mechanisms linking IgE sensitization and overweight/obesity to incident asthma through investigating DNAm during infancy ? a critical period of immune and lung development. The project will also provide a strong evidence base for developing targeted interventions for the primary prevention of childhood asthma.